Method for detecting biological toxins

ABSTRACT

Biological toxins are indirectly detected by using polymerase chain reaction to amplify unique nucleic acid sequences coding for the toxins or enzymes unique to toxin synthesis. Buffer, primers coding for the unique nucleic acid sequences and an amplifying enzyme are added to a sample suspected of containing the toxin. The mixture is then cycled thermally to exponentially amplify any of these unique nucleic acid sequences present in the sample. The amplified sequences can be detected by various means, including fluorescence. Detection of the amplified sequences is indicative of the presence of toxin in the original sample. By using more than one set of labeled primers, the method can be used to simultaneously detect several toxins in a sample.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to detecting biological toxins and, moreparticularly, to a method for amplifying genes coding for the toxins anddetecting the amplified genes, which detection is indicative of thepresence of the toxins.

2. Description of the Related Art

The ability to detect the presence or absence of biological toxins, suchas botulin (a toxin produced by the bacteria Clostrium botulinum) oraflatoxins (a toxin produced by the fungus Aspergillus favus)contaminating food, various biological warfare agents contaminating theair, and various contaminants of water or biological samples, has been adesire of scientists for ages. Such an ability would help preventdisease, incapacitation or other maladies attributable to the toxins,and would be useful in countering the use of biological weapons.

Current tests for detecting toxins usually depend on chromatographictechniques, such as gas or high-pressure-liquid chromatography, massspectroscopy, in vivo tests or in vitro bioassays. In vivo tests involveinjecting the toxin into animals, usually mice, at varying doses todetermine lethality. In vitro bioassays rely on the use of an antibody,receptor or living cell that binds the toxin directly. Thus, the actualtoxin is sought to be detected.

In other cases, assay methods are used to detect an organism which isthe source of the toxin. For example, if a pathogenic organism is knownto be associated directly with a toxin, tests have sought to detect thepresence of the pathogen. Such tests may include culturing the liveorganism.

The conventional tests described above generally are not very sensitive.They also may require pre-concentration of a sample suspected ofcontaining the toxin or organism. Further, in some cases, at leastpartial purification is necessary to remove interfering substances.These steps can be time-consuming and costly.

To date, there has not been developed a quick and efficient method fordetecting the presence of a biological toxin in a sample, said detectionbeing possible even if the toxin has been denatured or the organismresponsible for producing the toxin is no longer present.

SUMMARY OF THE INVENTION

Accordingly, it is a purpose of the present invention to provide a moresensitive method for detecting biological toxins.

It is another purpose of the present invention to detect toxins in thefood service, food preparation and dairy industries.

It is another purpose of the present invention to detect toxins used asoffensive biological weapons.

It is another purpose of the present invention to detect genes that havebeen willfully engineered into otherwise innocuous microorganisms(“weaponized organisms”).

It is another purpose of the present invention to provide a method fordetecting a biological toxin by testing for the presence of the uniquenucleic acid sequence responsible for the synthesis of the toxin or aspecific protein (enzyme) used to produce the toxin.

It is another purpose of the present invention to detect a plurality ofdifferent biological toxins in the same sample, simultaneously.

It is another purpose of the present invention to detect target nucleicacid sequences using a simple and easy to use method, which detection isindicative of the presence of toxins coded by the sequences.

It is another purpose of the present invention to solve the problemsassociated with conventional detection of low amounts of biologicaltoxins, of the biological entity responsible for producing the toxin,such as a microorganism, or of the nucleic acids coding for thesetoxins.

It is another purpose of the present invention to detect the presence inenvironmental and biological samples, of specific genes that encodetoxins, regardless of whether the gene is present in its normal genetichost, or whether it has been inserted into a different host by geneticengineering or by a random, natural gene transfer event.

It is another purpose of the present invention to provide a method foridentifying toxins in environmental or biological samples by detectingminute traces of genetic material that encode said toxins, and which arefound in association with said toxin preparations as a byproduct of thetoxin manufacturing process, such as toxins produced for disseminationas offensive biological weapons.

To achieve the foregoing and other purposes of the present inventionthere is provided the following method for detecting biological toxinsin samples. Instead of trying to directly detect the toxin present, thenucleic acid coding for the toxin is amplified using the known methodpolymerase chain reaction (“PCR”), and the amplified nucleic acid isthen detected as an indication of the presence or absence of the toxin.

More particularly, samples, such as clinical, food, water supply or airsamples, suspected of containing biological toxins are collected. Withthe unique gene sequence coding for the toxin being known, a primer paircan be selected which is complementary to the gene sequence. The primersand an appropriate enzyme for amplification are then mixed with thesample. The preparation is then placed into a thermal cycler, whereinPCR amplifies any toxin gene present. Thereafter, methods are used todetect the amplified gene. Detection of the gene coding for a toxin isindicative of the presence of the toxin in the original sample.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is known that large quantities of proteins and small molecules can beproduced by fermentation. For pharmaceutical applications, the productsare carefully purified and screened to remove residual DNA. On the otherhand, if the products are to be used for environmental applications,such as bioremediation or biological warfare, large quantities are madebut rigorous purification is not performed, because costs are too highand there is no need for purification. The DNA coding for the product orfor proteins (enzymes) that make the product remains with the product asa contaminant. The DNA is usually at too low a concentration to bedetected by conventional methods.

Methods are known for multiplying the DNA, such as the recombinant andcloning techniques discussed in U.S. Pat. Nos. 4,675,283, Roninson;4,925,792, Rappuoli; or 4,666,837 and Harford et al. However, suchmethods are not very fast, and the amount of the DNA produced still maynot be enough to ensure accurate detection of the genetic material.

Another available method of amplification is known as polymerase chainreaction (“PCR”). With PCR nucleic acid sequences can be quickly andexponentially amplified for facilitated detection of the sequences.

More particularly, as described in U.S. Pat. Nos. 4,683,202, Mullin;4,683,195, Mullis et al.; and 5,008,182, Sninsky et al.; as well asSaiki et al., Science, 230, 1350-1354 (1985); and Saiki et al., Science,239, 487-491 (1988), the disclosures of which are expressly incorporatedherein, PCR allows in vitro amplification of a nucleic acid sequencewhich lies between two regions of a known, longer sequence.

More particularly, double-stranded target nucleic acid is first meltedto separate the nucleic acid into the two strands to form templates, andthen oligonucleotide (“oligo”) primers complementary to the ends of thesegment which is desired to be amplified are separately annealed to thetemplates. The oligos serve as primers for the synthesis of newcomplementary strands, using a polymerase enzyme and a process known asprimer extension. The orientation of the primers with respect to oneanother is such that a 5′ to 3′ extension product from each primercontains, when extended far enough, the sequence which is complementaryto the other oligo. Thus, each newly synthesized nucleic acid strandbecomes a template for synthesis of another nucleic acid strandbeginning with the other oligo as a primer. Repeated cycles of melting,annealing of oligo primers, and primer extension lead to doubling, witheach cycle, of the strands containing the sequence of the templatebeginning with the sequence of one oligo and extending with the sequenceof the other oligo. The cycling is therefore exponential and after about20-30 cycles, the target nucleic acid can be amplified a million fold.

A critical difference between the present invention and existingPCR-based diagnostic assays is that the existing assays all seek toidentify the presence in a sample of the actual infectious organismitself. In these cases, it is the virus or bacteria, such as the HIVvirus, that is the causative agent sought to be detected. Some existingassays are described in U.S. Pat. No. 5,008,182 and PCT PublishedApplication No. WO 90/06376.

With the present invention, the organism itself is of no concern.Rather, the present invention focuses on a specific product, usuallyprotein based, produced by an organism. This product, a toxin, isencoded by a unique gene. The present invention seeks to identify thepresence of that unique gene in a sample, thereby strongly inferring thepresence of the product.

The source of the toxin can be animal, plant or microorganism. However,in all cases the target of the assay is a non-toxic contaminant (DNA orRNA) rather than the actual toxin product or the organism itself.

Moreover, as suggested above, existing rapid assays for toxins oftenemploy the strategy of antibody-based detection utilizing proven formatssuch as ELISA or RIA. These antibody-based systems employ no type ofamplification of nucleic acid sequences. Rather, their sensitivity andspecificity depend on highly restricted binding between antibody andtarget antigens (toxins). In this regard, the toxin products ofgenetically altered toxin genes, while retaining their toxic potential,may not be detectable by antibody assays developed for the unalteredtoxin. This is not the case for the assay according to the presentinvention, in which the gene, even if altered, is detectable using PCRtechnology, which requires significant, but not 100%, homology betweenthe priming sequences and only the ends or flanking regions of the DNAto be amplified.

Further, any test that concentrates on detecting an organism and not thetoxin per se may be rendered useless when the organism is no longerpresent or no longer viable. For example, it is frequently not anorganism per se in food that causes illness, but a toxin produced by theorganism. Testing for the organism, if the organism is not present ornot viable, may fail to identify the presence of the toxin. In contrast,with this invention, presence of the organism, such as Clostridiumbotulinum, is not needed in order to detect the presence of botulin;only the coincidental presence of the nucleic acid sequence responsiblefor coding the toxin is needed.

Thus, as described in detail below, the present invention utilizes PCRas a vehicle for quick and efficient amplification of geneticdeterminants of biological toxins, and then detects the amplifiedgenetic determinants as an indication of the presence or absence of thetoxins coded by the genetic material.

According to a preferred method of the present invention, a samplesuspected of containing a biological toxin is collected. By “toxin” itis meant a poisonous substance of biological origin, which necessarilyexcludes synthetic toxins which contain no contaminating DNA. The toxinsare usually, but not necessarily, proteins. Nonlimiting examples ofprotein toxins include botulin, perfringens toxin, mycotoxins,shigatoxins, staphylococcal enterotoxin B, tetanus, ricin, cholera,aflatoxins, diphtheria, T2, seguitoxin, saxitoxin, abrin, cyanoginosin,alphatoxin, tetrodotoxin, aconotoxin, snake venom, scorpion venom andother spider venoms. A nonlimiting example of a non-protein toxin istricothecene (T-2). Toxin-producing microorganisms of interest include,but are not limited to: Corynebacterium diphtheriae, Staphylococci,Salmonella typhimuium, Shigellae, Pseudomonas aeruginosa, Vibriocholerae, Clostridium botulinum, and Clostridium tetani. A nonlimitingexample of a toxin producing plant is Ricinus communis, and of a fungusproducing a toxin is Aspergillus favus.

Gene sequences coding for many toxins, such as botulin toxin types A-E,have been published and are available in a computer data base. If thesequence is not known, the organism responsible for producing the toxinis grown and the DNA removed and analyzed using known methods todetermine the appropriate sequence. Since the toxins are very dangerous,e.g. botulin, special precautions must be taken to protect laboratoryworkers and the general public.

The gene sequence must be specific to a particular organism or else thesequences of several organisms might be amplified using PCR. As aresult, once a sequence is detected, it must be checked against relatedorganisms incapable of producing the toxin of interest to ensure thatthe DNA of the related organism would not be amplified by PCR, or else anonspecific reading would occur.

With non-protein toxins, one looks for the gene that encodes an enzymethat is very specific for and critical to the production of the toxin.

The sample suspected of containing a toxin can be from any source. Forexample, an environmental (such as air, water, soil, surface swipes) orbiological (blood, mucous, saliva) sample can be used, provided itcontains or is suspected of containing the unique nucleic acid sequence,i.e., “target” nucleic acid, responsible for producing the toxin.

The sample is first processed, if necessary (centrifugation, addition oflysing agents), to obtain a crude preparation which contains at least afraction of any target nucleic acid that may be present in the sample.For example, before amplification the sample may be treated with anamount of a known reagent effective to open cells, etc., in the sample,and to expose the strands of the nucleic acids. These lysing anddenaturing steps allow amplification to occur much more readily.

If not already aqueous, an aqueous extract of the sample is prepared.

For the purpose of safety, a protein toxin can be denatured, forexample, by the addition of phenol. This treatment does not affect theDNA and the solution is centrifuged to separate the DNA portion.

It is also necessary to separate the strands before the genetic materialcan be used as a template, either as a separate step or simultaneouslywith the synthesis of the primer extension products. This strandseparation step can be accomplished by using any known suitabledenaturing conditions, including physical, chemical or enzymatic means.

The target nucleic acid sequence, i.e. the unique gene responsible forcoding for the toxin, which should be contained in the crudepreparation, is then amplified by PCR according to the following steps.

The preparation is mixed with an enzyme appropriate for PCR, i.e., anagent for polymerization, a pair of primers, an appropriate buffer(s),and deoxyribonucleotide triphosphates, each of which are described morefully below.

The “agent for polymerization” may be any compound or system, includingenzymes, which accomplishes the synthesis of primer extension products.Suitable enzymes for this purpose include, for example, Taq (Thermusaquaticus) polymerase, Q-beta replicase, E. coli DNA polymerase I,Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, otheravailable DNA polymerases, reverse transcriptases and other heat-stableenzymes.

The term “primer” as used herein refers to a molecule, comprised of twoor more deoxyribonucleotides or ribonucleotides, which is capable ofacting as a point for initiating synthesis when placed under conditionswhich induce the synthesis of a primer extension product substantiallycomplementary to a nucleic acid strand, i.e., in the presence of thedeoxyribonucleotide triphosphates and the agent for polymerization andat the suitable temperature and pH. Preferably, the primer is anoligodeoxyribonucleotide.

The primer is preferably single stranded for maximum efficiency inamplification, but may alternatively be double stranded. If doublestranded, the primer is first treated to separate its strands beforebeing used to prepare extension products.

The primer's sequence must be complementary to the unique target nucleicacid sequence which is responsible for coding the toxin of interest andappropriate to the goals of the assay, i.e. the primer should be chosenso that the target DNA amplified would be selected with appropriatespecificity. In this regard, the primer must be sufficiently long andspecific enough to prime the synthesis of the extension products in thepresence of the agent for polymerization. Statistically speaking, theprimer is preferably at least 12 bases long, to ensure the primerdescribes a sequence that is unique to that organism. Further, becausemany organisms share some common conserved sequences, the primer shouldbe specific to the particular type of organism responsible for producingthe toxin, and not a closely related but non-toxin producing type.

With the present invention, one can dictate the level of specificity ofthe assay. One can use primers which will identify toxin producing genesfrom any of several organisms, by targeting common nucleic acidsequences. For example, if one wanted to detect all Clostridiumbotulinum types in a single sample, e.g. types A-F, theoretically, aprimer could be prepared, if the types A-F shared a common nucleic acidsequence responsible for producing the botulin toxin. However, it is notcurrently known whether such a common nucleic acid sequence exists amongClostridium botulinum. A given toxin gene can also be among Clostridiumbotulinum exists. A given toxin gene can also be shared by more than oneorganism. For example, the heat-labile toxin gene is shared by bothVibrio cholerae and toxigenic strains of Escherichia coli. Toxin genestransferred into non-native host organisms could also be detected usingthe method of the present invention. Thus, the primer sequence can bedetermined and a primer prepared, once the unique sequence of the targetnucleic acid is determined. The exact lengths of the primers will dependon many factors, including temperature, buffer, nucleotide compositionand source of primer.

PCR occurs in a buffered aqueous solution, preferably at a pH of 7-9,most preferably about 8.

The deoxyribonucleotide triphosphates dATP, dCTP, dGTP and/or TTP arealso added to the synthesis mixture, either separately or together withthe primers, in adequate amounts and the resulting solution is heated ina thermal cycler according to the following sequence.

1. A high temperature (usually about 94° C.), at which the two strandsof the double stranded target melt, or denature, and separate intosingle strands.

2. A low temperature (37-54° C.), at which the primers bind (anneal) totheir homologous regions on the single stranded target.

3. An intermediate temperature (about 72° C.) at which the agent forpolymerization, e.g. Taq polymerase, binds to the primer complex andmoves down the single stranded target, copying it and making a duplicatesecond strand.

The exact temperatures for each of the steps must be determinedempirically for the particular target to be amplified, although optimumconditions can be estimated, based on certain characteristics of thetarget.

It is also possible to carry out a two-step temperature cycle whereinthe third above-described step is eliminated. This is because in heatingfrom the second step directly back to the first step the reaction passesthrough the mid-range over a period of time which, although brief, canbe sufficient to allow complete primer extension to occur.

This thermal cycle is then repeated until the target DNA has beenamplified sufficiently to permit its detection. Typically, the thermalcycle is repeated about 20 to 30 times, resulting in an approximatelyone million-fold amplification of the target DNA.

A second amplification step can be followed, wherein a second set ofprimers can introduce a detection label, such as biotin, “doublestranded DNA binding protein” (dsDNA-BP) binding sites, or fluorophoresinto the amplified genes. See Kemp et al., Proc. Natl. Acad. Sci., pp.2423-2427 (1989) and co-pending, co-assigned U.S. patent applicationSer. No. 07/635,019, filed Dec. 28, 1990, in the name of one of theinventors herein, Dr. James Campbell, the subject matter of which isexpressly incorporated by reference herein). These labels furtherfacilitate the detection of the amplified nucleic acid sequence, asdescribed below.

According to the teachings of the above-mentioned U.S. Ser. No. 635,019,samples suspected of containing microorganisms or characteristic DNA areprocessed as appropriate (centrifugation, addition of lysing agents, orno processing) to obtain a crude preparation which contains at least afraction of any target DNA that may be present in the sample. The samplecan be from any source. For example, the sample can be environmental(such as air, water, soil, surface swipes) or biological (blood, urine,feces, etc.).

The target DNA from the crude preparation is then amplified by thepolymerase chain reaction. The polymerase chain reaction is a well-knowngeneralized procedure for amplifying target DNA. The polymerase chainreaction is described in Saiki et al, Science, 230, 1350-1354 (1985),and Saiki et al, Science, 239, 487-491 (1988), both of which areincorporated herein by reference. In general, a sample of the crudepreparation is added to an aqueous solution containing an enzymeappropriate for PCR (such as Taq polymerase or Q-beta replicase)appropriate buffers, deoxyribonucleotide triphosphates, and two primers.

The thus prepared reaction mixture is then thermally cycled at threesuccessive temperatures:

1. A high temperature (usually about 94° C.), at which the two strandsof the double stranded target DNA melt, or denature, and separate intosingle strands.

2. A low temperature (37-54° C.), at which the primers bind (anneal) totheir homologous regions on the single stranded target DNA.

3. An intermediate temperature (about 72° C.) at which the Taqpolymerase binds to the DNA-primer complex and moves down the singlestranded target, copying it and making a duplicate second strand. Theexact temperatures for each of the steps must be determined empiricallyfor the particular DNA to be amplified, although optimum conditions canbe estimated, based on certain characteristics of the DNA. It is alsopossible to carry out a two-step temperature cycle wherein the third(72° C.) step described herein is eliminated. This is because in heatingfrom the second (37-54° C.) step directly back to the first (94° C.)step the reaction passes through the 72° C. range over a period of timewhich, although brief, can be sufficient to allow complete primerextension to occur.

This thermal cycle is then repeated until the target DNA has beenamplified sufficiently to permit its detection. Typically, the thermalcycle is repeated about 20 to 30 times, resulting in an approximatelyone million-fold amplification of the target DNA.

The thus amplified target DNA is then subjected to a second, brieferamplification. This second amplification uses a second pair of primersthat are nested within the region bracketed by the first set of primers.In other words, the second pair of primers is complementary to, andbinds with, sites on the amplified DNA within the region bracketed bythe first set of primers.

The second primers also differ from the first primers in that at leastone member of the second pair has attached covalently to its 5′ end, afluorescent reporter molecule, (e.g., FITC, TRITC, Rhodamine, TexasRed). In addition, at least one member of the second primer pair hascovalently attached to its 5′ end a particular DNA fragment thatconstitutes a specific binding site for a double-stranded DNA bindingprotein (dsDNA-BP). Typically, the dsDNA-BP is attached to one member ofthe primer pair and the FRM is attached to the other member of theprimer pair. Nevertheless, it is possible to covalently bind both thedsDNA-BP binding site and the FRM to only one of the primers and leavethe other primer unmodified. This is accomplished by designing oneprimer with the dsDNA-BP binding site covalently attached to the 5′ endof the primer, and the FRM covalently attached to the 5′ end of thebinding site. In an evanescent wave detection format, this modificationmay result in greater sensitivity of the system by bringing the FRMcloser to the surface of the optical fiber, for more efficientexcitation and more efficient coupling of emitted light back into thefiber. The second amplification is typically shorter than the firstamplification and need only be sufficient to ensure that theconcentration of twice-amplified DNA, which contains the fluorescentreporter molecule and the double stranded DNA-binding protein, will emita detectable level of fluorescence energy when the DNA is bound to thesensor and exited by light of an appropriate wavelength.

A dsDNA-BP is attached to a fluorescent sensor by any means appropriatefor fixing a protein to the exposed portion of the material of thesensor. Several techniques have been used to attach proteins to varioussubstrates and these techniques may also be useful in the presentinvention. Typically, the substrate for the dsDNA-BP is made of glass ormembrane material and the dsDNA-BP is attached using a techniqueappropriate for the fixing of a protein to glass or synthetic membranessuch as nitrocellulose or PVDF. One technique for immobilizing proteinon glass which is particularly useful in the present invention isdescribed by Bhatia et al “Fiber Optic-based Immunosensors: A ProgressReport”, SPIE vol. 1054 Fluorescence Detection III (1989).

The exposed surface of the sensor having the dsDNA-BP immobilizedthereon is then exposed to the twice amplified DNA preparation, which,if it originally contained the target DNA, now contains the amplifiedtarget DNA with reporter molecules and dsDNA-BP binding site attached.Any target DNA amplified with the second set of primers will then bind,via the dsDNA binding site, to the dsDNA-BP immobilized on the exposedsurface of the sensor. If the sensor is an optical waveguide, theevanescent wave excites the fluorescent reporter molecule, which hasbeen positioned in close proximity to the outer surface of the sensorvia binding of the dsDNA-BP with the dsDNA-BP binding site on the DNA.The fluorescence of the excited fluorescent reporter molecule, at awavelength different from that of the excitation energy, is also readilycoupled back into the sensor, which detects the fluorescent emission byknown means. If the exposed surface of the sensor is a membrane, thedsDNA-BP is attached to the membrane either by adsorption or by covalentlinkage, and unreacted sites on the membrane are blocked by any ofseveral blocking reagents. The twice-amplified DNA is then applied tothe membrane, where it binds to the attached protein via the bindingsite incorporated into the amplified DNA. The membrane is thenintroduced into a fluorometer, typically via a dipstick or slide device,and fluorescence intensity measured.

Several dsDNA-BP's are known to exist and can be used according to thepresent invention.

A fluorescent sensor useful according to the present invention can beany one having an exposed surface of a material onto which a dsDNA-BPcan be immobilized. Typically, the sensor will have an exposed surfacemade of a silica-based glass or a plastic, but other materials such asnitrocellulose or PVDF membrane may also be used. The geometry of thewaveguide, membrane, or other surface is not critical to the presentinvention.

Because the primer pair for at least the first amplification step isselected to be homologous to base sequences unique to the target nucleicacid, only the target nucleic acid is amplified. Because the targetnucleic acid is greatly amplified, the amplified target becomesessentially the only nucleic acid in the sample preparation after thefirst amplification step has been completed. Thus, spuriousamplification of nucleic acid other than target nucleic acid in anysecond amplification step is essentially avoided.

Moreover, as individual primers are “invisible” to and do not interactwith each other, several different sets of primers can be usedsimultaneously in the same reaction tube to probe a sample for multiplenucleic acid sequences associated with a number of different biologicaltoxins. By attaching different detection labels to the different primersets, the presence of different nucleic acids can be determined. Thistechnique provides a unique capability for simultaneous multi-toxindetection, which is not available in conventional detection methods.

A variety of tests can be used to identify the amplified nucleic acidsequences. These tests include dot blots, Southern blots, polyacrylamideor agarose gel electrophoresis, calorimetry, fluorescence, membraneaffinity filtration and identification, and differential affinitychromatography, each of which is described below.

In the dot blot method, the amplified sample is spotted directly on amembrane and labeled with a probe. The probe can be an enzyme such asalkaline phosphatase, a radioactive label such as ³²P, a fluorescentlabel, or biotin. The labelled probe may be detected by spectroscopy,photochemistry or by biochemical, immunochemical or chemical means.

In Southern blots, gel electrophoresis separates DNA fragments intotheir size classes. The fragments are then denatured by placing the gelin alkali after which a nitrocellulose filter sheet is laid over thegel. The DNA fragments are transferred from the gel to a filter byplacing layers of absorbent paper above the latter (blotting), with thegel being immersed in a high concentration of salt. This causes thesolute and DNA fragments to diffuse through the nitrocellulose sheet towhich the latter are adsorbed, maintaining their relative positions. Thenitrocellulose sheet is then incubated under renaturing conditions witha solution containing radiolabelled molecules able to base-pair withsome sequence in the original DNA (probing). Finally, the position andtherefore fragment size of the complementary sequence in the originalDNA can be located by autoradiography or fluorescence. Chambers BiologyDictionary, Walker, ed., W. & R. Chambers Ltd. and Cambridge UniversityPress, 1989.

The polyacrylamide or agarose gel method is used for separating nucleicacids or proteins on the basis of charge, shape and size. The highlycross-linked polymer of acrylamide or agarose forms a transparent gelmatrix through which macromolecules move under the influence of anelectric or magnetic field, resulting in discrete bands of DNA. Id.

The fluorescence method is described in the above-cited co-pending U.S.patent application No. 07/635,019. In this method, the second set ofprimers is modified so that at least one contains a specific bindingsite for a dsDNA-BP and at least one has a fluorescent reporter molecule(FRM) covalently attached thereto. The dsDNA-BP is attached to thesurface of a fluorometric device by adsorption or covalent interaction.In an assay, the amplification of the target DNA with concomitantinclusion of both the FRM and dsDNA-BP binding site, makes the amplifiedportion the only DNA detectable by the fluorometric device, such as afluorescence sensor. The amplified target DNA, with the FRM and thedsDNA-BP binding site attached thereto, specifically binds with thedsDNA-BP on the fluorometric device. This binding brings the FRM closeto the surface of the fluorescent fluorometric device, where it can beexcited. For fluorometric devices such as evanescent wave-based systems,the close proximity of the FRM to the surface of the fluorometric deviceenhances both the excitation of the FRM, as well as the coupling of theenergy emitted by the excited FRM at a wavelength different than that ofthe excitation energy) back into the fluorometric device. Forfluorometric devices whose exposed binding surface employs a membrane,e.g., in dipstick or slide format, the binding of the fluorescent DNA bythe dsDNA-BP permits it to be manipulated in separation steps or forintroduction into fluorometric devices. At the photodetector, the signalis discriminated and interpreted by a microprocessor. The entireexcitation, emission, detection and interpretation process can occur inseconds.

Membrane affinity filtration and identification involves a membraneattached to an absorptive pad. A “capture reagent”, e.g., a dsDNA-BP, isadsorbed to the membrane. The PCR-amplified target DNA, which has on oneend a binding site for the dsDNA-BP and on the other end a biotinmolecule, is allowed to react 5 minutes in a small test tube with anequal volume of a “color reagent.” The color reagent may consist, forexample, of streptavidin (a molecule that binds with high affinity tobiotin) bound to either colloidal gold, or to a fluorophore. Thismixture is then applied to the membrane, where it is rapidly (10seconds) drawn through by capillary action into the absorptive padbelow. The complex is captured via its binding site by the dsDNA-BP. Inthe case of the colloidal gold label, a positive result is indicated bythe immediate appearance of a red spot on the membrane. For thefluorophore label, the membrane is introduced into a fluorometer tomeasure the fluorescence signal.

Differential affinity chromatography utilizes a membrane or other solidsupport, such as a commercially available syringe filter unit (i.e.,ACTI-DISK®) through which is treated with the PCR product. In the caseof the filter, typically a filter disk, is prepared by a two-stepprocedure: First the dsDNA-BP is covalently bound to the disk. Next, allthe binding sites of the dsDNA-BP are occupied by exposure to anextraneous PCR product containing a “low-affinity” binding site for thedsDNA-BP. The “normal” binding site for the dsDNA-BP consists of aspecific 10 nucleotide sequence of DNA, whereas the “low-affinity”binding site consists of this 10 nucleotide sequence in which one ormore of the nucleotides has been substituted with a different one, toreduce the overall affinity of the product for the dsDNA-BP bindingsite. This low affinity PCR product also has attached to its other endan enzyme such as horseradish peroxidase or alkaline phosphatase oralternatively, a fluorophore. When the PCR product containing the targetDNA with the “normal” binding site is introduced into the filter disk,it easily out-competes the previously bound low-affinity product, whichelutes off the membrane and falls into a small container including thesubstrate for the horseradish peroxidase or alkaline phosphatase,causing a color to develop. In the case of a fluorophore label, theeluted product falls directly into the detection chamber of afluorometer for readout. The reagents are introduced to the filter diskeither by a syringe, or by continuous pumping through tubing. The lattertechnique allows a continuous, flow-through monitoring of PCR samples asthey are produced.

Without further elaboration, it is believed that one skilled in the art,using the preceding description, can utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limiting inany way whatsoever, or of the remainder of the disclosure.

The following various experiments demonstrate that a toxin can bedetected by identifying in a sample the DNA that encodes the toxin. Theparticular examples include amplification of the toxin genes for severalstrains of Clostridium botulinum. Samples tested included both crudebacterial lysates, provided by Fort Detrick, Md., as well as purifiedtoxin protein preparations purchased from SIGMA Chemicals. Such purifiedpreparations are also available from Wako Chemicals.

EXAMPLES Example 1

A crude, unpurified type D Clostridium botulinum (BotD) toxinpreparation, and a DNA extract of the same crude toxin preparation, wereselected as samples. Each was amplified and detected separately. Thenucleic acid sequence coding for this toxin is known.

Ten μl of each BotD DNA sample (0.5 mg./0.5 ml 0.2 M NaCl, 0.05 M Naacetate, pH 6.0) was prepared as follows:

0.5 ml toxin preparation was added to an equal volume of saturatedphenol. The resultant mixture was vortexed briefly, and centrifuged for5 min. at 14,000×g. The supernatant was recovered and the phenolextraction step was repeated on the recovered supernatant. An equalvolume of chloroform was added to the supernatant recovered from therepeated phenol extraction. The supernatant was removed and chloroformextraction repeated. The DNA was then precipitated as a pellet fromsupernatant ethanol. The pellet was then resuspended in 50 μl distilledH₂O.

The preparation was added to the conventional PCR reagents Taqpolymerase, a buffer, and a mixture of deoxyribonucleotidetriphosphates. The following pair of oligonucleotide primers was alsoadded to the solution:

BotD1 5′-TGA CAT GGC CAG TAA AAG-3′ (SEQ ID NO: 1)

BotD2 5′-TGT TCC AAA CCC TTC AAA-3′ (SEQ ID NO: 2)

These primers were designed by co-inventor James Campbell, of the NavalResearch Laboratory, and synthesized by the Synthecell Corporation ofGaithersburg, Md. These primers are specific oligonucleotide pairsdesigned to match unique DNA sequences in the gene encoding type Dbotulin toxin.

The thus prepared reaction mixture was used in PCR by thermally cyclingfor two minutes at three successive temperatures.

1. A high “denaturing” temperature (94° C.);

2. A low “annealing” temperature (52° C.);

3. An intermediate “extension” temperature (about 72° C.).

This thermal cycle was repeated 30 times so that the target DNA wasamplified sufficiently to permit its detection, resulting in anapproximately one million-fold amplification of the target DNA.

The PCR product was examined by electrophoresing a small portion thereofthrough an agarose gel, staining with ethidium bromide, and viewing thestained gel under U.V. light. Since it is known in advance what sizepiece of the toxin gene was bracketed by the two primers, a positiveresult was indicated for each sample by the appearance of a singlestained band of DNA of the appropriate size, at the predicted M.W. (500bp).

Example 2

A crude, unpurified type A Clostridium botulinum (BotA) toxinpreparation, and a DNA extract of the same crude toxin preparation, wereselected as samples. Each sample was amplified and analyzed separately.The nucleic acid sequence coding for this toxin is known.

Ten μl of the preparation made according to the steps set out inExperiment 1 was added to an aqueous solution including the sameconventional PCR reagents as in Example 1, except that the primer pairconsisted of BotA1 and BotA3. These primers were designed by co-inventorJames Campbell to match unique DNA sequences in the gene encoding type Abotulism toxins, and synthesized by the Synthecell Corporation. Primersequences are as follows:

Bot A1 5′-ATT AAT TAT AAA GAT CCT-3′ (SEQ ID NO: 3)

Bot A3 5′-AAC TTC AAG TGA CTC CTC-3′ (SEQ ID NO: 4)

The thus prepared reaction mixture was cycled thermally for two minutesat three successive temperatures.

1. A high “denaturing” temperature (94° C.);

2. A low “annealing” temperature (52° C.); and

3. An intermediate “extension” temperature (72° C.).

This thermal cycle was repeated 30 times.

The PCR product was examined by electrophoresing a small portion thereofthrough an agarose gel, staining with ethidium bromide, and viewing thestained gel under U.V. light. Since it is known in advance what sizepiece of the toxin gene was bracketed by the two primers, a positiveresult was indicated for each sample by the appearance of a singlestained band of DNA which is of the appropriate size at the predictedM.W. (585 bp).

The same method has been used to detect the botulinum toxin produced bythe remaining Clostrium botulinum strains B, C, E and F, as well asother toxins by using appropriate PCR primers.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

The present invention's use of genetic material to identify a proteintoxin provides an alternative (second) technology for verification ofthe above-described conventional methods that depend on antibody orprotein analysis.

As described above, the present invention relies on the principle thatPCR provides an efficient and quick mechanism to amplify contaminatingnucleic acid sequences. While the actual concentration of the relevantnucleic acid sequence in the sample may be as low as 1/1000 of the toxinconcentration, increasing the concentration of the sequence amillion-fold prior to analysis greatly facilitates detection of thesequence. Methods which can screen for the characteristic sequence afteramplification are then more successful in indicating the presence ofbiological toxins than testing directly for the biological toxin itself.

The ultimate detection sensitivity level of the present invention is notyet known. However, the minute amount of toxin gene DNA found as acontaminant in as little as 100 picograms of protein could bespecifically detected.

The selection of target nucleic acid for amplification, rather than thedetection of the microorganism making a toxin may permit the presentinvention to detect toxins that have been genetically engineered. Forexample, the present invention detects the presence of the botulin genein any organism, thus indicating that the organism is potentiallycapable of producing the botulin toxin, regardless of whether the genefor that toxin was naturally present in the organism or whether the genewas inserted into the organism by genetic manipulation.

As shown above, the present invention is even effective if the toxin hasbecome denatured. That is, the residual DNA is still present and can beamplified, regardless of the condition of the toxin. This providesdistinct benefits over conventional methods. That is, the presentinvention makes testing far more reliable than with the conventionalmethods testing for the toxins per se, which fail if the toxin isdenatured. Further, with the conventional tests, special precautionsmust be taken to protect laboratory workers and prevent escape of thetoxin into the environment. The precautions usually require speciallyequipped, expensive laboratories and sometimes government clearance.With the present invention, the toxin can be rendered harmless beforetesting, but its presence can still be detected based on the residualDNA. As a result, testing can be performed less expensively, in morelaboratories, and more safely.

Further, the present invention basically requires only a commerciallyavailable thermal cycler, PCR reagents and a readout device to performthe amplification and detection. As such, a small transportable systemcan be used in the field to carry out the present invention. The systemis not envisioned as something to be carried around by soldiers incombat to detect biological warfare agents. However, the system could belocated in a region of conflict. Other practical uses of the systeminclude: detection of the presence of toxin contamination of a watersupply; testing commercial toxin preparations for the presence of DNA,even though these preparations are supposedly purified; detection of thepresence of toxins in food or beverages; and detection of the presenceof biological toxins at sites which are under review by an internationalinvestigative or oversight body.

The foregoing is considered illustrative only of the principles of theinvention. Further, since numerous modifications and changes willreadily occur to those skilled in the art, it is not desired to limitthe invention to the exact construction and operation shown anddescribed. Accordingly, all suitable modifications and equivalents maybe resorted to that fall within the scope of the invention and theappended claims.

4 18 base pairs nucleic acid single stranded circular Other nucleic acidno no not provided 1 TGACATGGCC AGTAAAAG 18 18 base pairs nucleic acidsingle stranded circular Other nucleic acid no no not provided 2TGTTCCAAAC CCTTCAAA 18 18 base pairs nucleic acid single strandedcircular Other nucleic acid no no not provided 3 ATTAATTATA AAGATCCT 1818 base pairs nucleic acid single stranded circular Other nucleic acidno no not provided 4 AACTTCAAGT GACTCCTC 18

What is claimed is:
 1. A method for detecting a DNA coding for a toxinof biological origin or a DNA coding for an enzyme specific to toxinproduction, said toxin suspected of being present in a sample known notto contain any organisms which produce said toxin, comprising the stepsof: a) performing an amplification on said sample which is suspected ofcontaining a unique DNA having a unique sequence of nucleotides codingfor the toxin or for an enzyme specific to toxin production, said samplenot containing any organisms which produce said toxin, by initiating apolymerase chain reaction with a pair of primers complementary to saidends of said sequence of nucleotides and a polymerase which catalyzessaid polymerase chain reaction, said amplification being sufficientlygreat that the amount of DNA other than said unique DNA in the amplifiedsample is negligible in comparison to the amount of said unique DNA inthe amplified sample if said unique DNA is present in the unamplifiedsample; and b) detecting the presence of amplified unique DNA in saidamplified sample.
 2. The method as recited in claim 1, wherein the toxinis selected from the group consisting of botulin, tetanus, ricin,cholera, diphtheria, aflatoxins, perfringens toxin, mycotoxins,shigatoxin, staphylococcal enterotoxin B, T2, seguitoxin, saxitoxin,abrin, cyanoginosin, alphatoxin, tetrodotoxin, aconotoxin, snake venomand spider venom.
 3. The method as recited in claim 1, furthercomprising the step of: performing a further amplification on saidamplified sample by initiating a polymerase chain reaction with a pairof primers complementary to a unique sequence of nucleotides within saidamplified unique sequence of nucleotides and a polymerase whichcatalyzes said polymerase chain reaction, at least one member of saidsecond pair of primers having a detection label bound thereto, therebyproducing a detectable amount of double stranded, twice-amplified DNAwith said detection label on at least one end thereof if said toxin ispresent in the unamplified sample.
 4. The method as recited in claim 3,wherein said detection of the presence of amplified unique DNA in saidamplified sample in step (b) comprises the steps of: (1) spotting saidtwice-amplified sample directly on a membrane, and (2) detecting anylabelled DNA using one of spectroscopic, photochemical, biochemical orimmunochemical techniques.
 5. The method as recited in claim 1, whereinsaid detection of the presence of amplified unique DNA in said amplifiedsample in step (b) comprises the steps of: (1) using gel electrophoresisto separate any DNAs in the sample; (2) denaturing the DNAs; (3)adsorbing the denatured DNAs on a nitrocellulose membrane; (4)incubating the nitrocellulose membrane under renaturing conditions witha solution containing fluorescent or radio-labelled molecules able tobase pair with said unique DNA; and (5) detecting the position and sizeof the labelled, unique DNA using autoradiography or fluorometry.
 6. Themethod as recited in claim 1, wherein said detection of the presence ofamplified unique DNA in said amplified sample in step (b) comprises thesteps of: placing any amplified DNA in a matrix of agarose through whichsaid amplified DNA moves under the influence of an electric or magneticfield; and identifying said unique DNA based on its mobility in saidmatrix.
 7. The method as described in claim 3, wherein at least one ofsaid primers used in said further amplification includes a specificbinding site for a double-stranded DNA binding protein, and wherein anyof said twice-amplified DNA present includes said specific binding siteat one end and a biotin molecule at its other end.
 8. The method asrecited in claim 7, wherein said detection of the presence of amplifiedunique DNA in said amplified sample in step (b) comprises the step of:(1) using a capture reagent on a membrane attached to an absorptive pad;(2) reacting any of said twice-amplified DNA, including said doublestranded DNA binding site and said biotin molecule, with a color reagentto form a complex; (3) applying the complex to a membrane attached to anabsorptive pad, said membrane having bound thereto a double-strandedDNA-binding protein, whereupon the mixture is captured on the membrane;(4) detecting the color on the membrane.
 9. The method as recited inclaim 7, wherein said detection of the presence of amplified unique DNAin said sample in step (b) comprises the steps of: (1) using gelelectrophoresis to separate any DNAs in the sample; (2) denaturing theDNAs; (3) adsorbing the denatured DNAs on a nitrocellulose membrane; (4)incubating the nitrocellulose membrane under renaturing conditions witha solution containing fluorescent or radio-labelled molecules able tobase pair with said unique DNA; and (5) detecting the position and sizeof the labelled, unique DNA using autoradiography or fluorometry. 10.The method as recited in claim 3, wherein the detection label isselected from the group consisting of biotin, fluorophores andchromophores.
 11. A method for detecting a plurality of DNAs coding fortoxins of biological origin or DNAs coding for enzymes specific toproduction of said toxins in a sample suspected of including at leastone of said toxins and known not to contain any organisms which producesaid at least one toxin, comprising the steps of: a) performing anamplification on said sample which is suspected of containing aplurality of unique DNAs each having a unique sequence of nucleotidescoding for a respective one of said toxins or for an enzyme specific toproduction of a respective one of said toxins, said sample notcontaining any organisms which produce said toxin, by initiating apolymerase chain reaction with a plurality of pairs of primers, eachpair of said plurality of primers being complementary to said ends of arespective one of said sequences of nucleotides, and a polymerase whichcatalyzes said polymerase chain reaction, said amplification beingsufficiently great that the amount of DNA other than said unique DNAs inthe amplified sample is negligible in comparison to the amount of saidunique DNA in the amplified sample if any of said unique DNAs is presentin the unamplified sample; and b) detecting the presence of any of saidamplified unique DNAs in said amplified sample.
 12. The method asrecited in claim 11, further comprising the step of: performing afurther amplification on said amplified sample by initiating apolymerase chain reaction with a plurality of pair of primers, each pairof primers being complementary to a unique sequence of nucleotideswithin a respective one of said unique sequences of nucleotides and apolymerase which catalyzes said polymerase chain reaction, at least onemember of each of said second pairs of primers having a detection labelbound thereto, thereby producing a detectable amount of double stranded,labelled, twice-amplified DNA if any one of said toxins is present inthe unamplified sample.
 13. The method as recited in claim 12, whereinthe label associated with each primer pair is unique to said primerpair, thus permitting specific identification of the particularnucleotide sequence amplified.
 14. The method as recited in claim 12,wherein said detection of the presence of amplified unique DNA in saidamplified sample in step (b) comprises the steps of: (1) spotting saidtwice-amplified sample directly on a membrane, and (2) detecting any ofsaid labelled DNAs present on said membrane using one of spectroscopic,photochemical, biochemical or immunochemical means.
 15. The method asrecited in claim 11, wherein said detection of the presence of amplifiedunique DNA in said amplified sample in step (b) comprises the steps of:(1) using gel electrophoresis to separate any DNAs in the sample; (2)denaturing the DNAs; (3) adsorbing the denatured DNAs on anitrocellulose membrane; (4) incubating the nitrocellulose membraneunder renaturing conditions with a solution containing fluorescent orradio-labelled molecules able to base pair with said unique DNAs; and(5) detecting the position and size of any of said labelled, unique DNAspresent on said membrane using autoradiography or fluorometry.
 16. Themethod as recited in claim 11, wherein said detection of the presence ofamplified unique DNA in said amplified sample in step (b) comprises thesteps of; placing any amplified DNA in a matrix of agarose through whichsaid amplified DNA moves under the influence of an electric or magneticfield; and identifying said unique DNA based on its mobility in saidmatrix.
 17. The method as described in claim 12, wherein at least onemember of each of said primer pairs used in said further amplificationincludes a specific binding site for a double-stranded DNA bindingprotein, and wherein all of said twice-amplified DNAs present includesaid specific binding site at one end and a biotin molecule or afluorophore at its other end.
 18. The method as recited in claim 17,wherein said detection of the presence of amplified unique DNA in saidamplified sample in step (b) comprises the step of: (1) using a capturereagent on a membrane attached to an absorptive pad; (2) reacting any ofsaid twice-amplified DNAs present, including said double stranded DNAbinding sites and said biotin molecules, with a color reagent to form acomplex; (3) applying the complex to a membrane attached to anabsorptive pad, said membrane having bound thereto a double-strandedDNA-binding protein, whereupon the complex is captured on the membrane;and (4) detecting the color on the membrane.
 19. The method as recitedin claim 17, wherein said detection of the presence of amplified uniqueDNA in said amplified sample in step (b) comprises the steps of: (1)binding a double-stranded DNA-binding protein to a solid support; (2)occupying all double-stranded DNA-binding sites of said protein on saidsolid support by treating said support with a color indicator whichoccupies said binding sites of said protein but has a significantlylower affinity for said binding sites of said protein than do any ofsaid twice-amplified DNAs present in the sample; (3) treating the solidsupport with the twice-amplified sample amplified sequences having anormal affinity for said binding sites whereupon any of thetwice-amplified DNAs present in said twice-amplified sample displacessaid indicator from said support; and (4) colorimetrically orfluorometrically detecting the amount of said indicator displaced fromsaid support.
 20. The method as recited in claim 1, wherein said sampleis an environmental sample, water supply sample, purified commercialtoxin preparation, food or beverage sample, blood sample, mucous sample,saliva sample and urine sample.
 21. The method as recited in claim 20,wherein said environmental sample is an air, soil, surface swipe, orwater sample.
 22. The method as recited in claim 11, wherein said sampleis an environmental sample, water supply sample, purified commercialtoxin preparation, food or beverage sample, blood sample, mucous sample,saliva sample and urine sample.
 23. The method as recited in claim 22,wherein said environmental sample is an air, soil, surface swipe, orwater sample.
 24. The method as recited in claim 1, wherein said methodconsists essentially of steps (a) and (b).
 25. The method as recited inclaim 11, said method consists essentially of steps (a) and (b).